New GJA8 Variants and Phenotypes Highlight Its Critical Role in a Broad Spectrum of Eye Anomalies

Human Genetics (2019) 138:1027–1042 https://doi.org/10.1007/s00439-018-1875-2

ORIGINAL INVESTIGATION

New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies

Fabiola Ceroni1 · Domingo Aguilera‑Garcia2,3 · Nicolas Chassaing4,5 · Dorine Arjanne Bax1 · Fiona Blanco‑Kelly2,3,6,7 · Patricia Ramos2,3 · Maria Tarilonte2,3 · Cristina Villaverde2,3 · Luciana Rodrigues Jacy da Silva2,3 · Maria Juliana Ballesta‑Martínez8 · Maria Jose Sanchez‑Soler8 · Richard James Holt1 · Lisa Cooper‑Charles9 · Jonathan Bruty9 · Yvonne Wallis9 · Dominic McMullan9 · Jonathan Hofman10 · David Bunyan11 · Alison Stewart12 · Helen Stewart6 · Katherine Lachlan13,14 · DDD Study15 · Alan Fryer16 · Victoria McKay16 · Joëlle Roume17 · Pascal Dureau18 · Anand Saggar19 · Michael Grifths9 · Patrick Calvas4,5 · Carmen Ayuso2,3 · Marta Corton2,3 · Nicola K Ragge1,10

Received: 22 November 2017 / Accepted: 9 February 2018 / Published online: 20 February 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identifed four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identifed in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical signifcance were also identifed. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identifed heterozygous 1q21 microdeletions in fve families with coloboma and other ocular and/or extraocular fndings. However, the exact genotype–phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confrming the importance of this gene in early eye development.

Introduction anomalies, such as early onset cataract and anterior segment dysgenesis (ASD). They are associated with extraocular fea- Anophthalmia (absent eye), microphthalmia (small eye) tures in just over half of cases (Shah et al. 2012) and can and coloboma (optic fssure closure defects), collectively form part of a syndrome (Slavotinek 2011). The etiology referred to as AMC, form a spectrum of developmental of AMC is characterised by marked genetic heterogeneity. eye disorders, with an overall estimated incidence of 6–13 This refects the complexity underlying eye morphogenesis, per 100,000 births (Shah et al. 2011; Skalicky et al. 2013). a conserved process that requires a series of highly coordi- AMC can occur alone or in combination with other ocular nated events, both at the molecular and the structural level, and is tightly regulated by a network of transcription factors, extracellular signaling molecules, cell-cycle regulators, and Marta Corton and Nicola Ragge: joint senior authors. adhesion proteins (Reis and Semina 2015). Electronic supplementary material The online version of this Connexins (Cxs) are a homogeneous family of transmem- article (https://doi.org/10.1007/s00439-018-1875-2) contains brane proteins with a crucial role in intercellular communi- supplementary material, which is available to authorized users. cation. They present a conserved topology, which consists of four transmembrane α-helices (TM1–TM4) joined by * Nicola K Ragge [email protected]; [email protected] two extracellular loops (ECL1 and ECL2) and one cytoplasmic loop (ICL), fanked by a short cytoplasmic N-terminal Extended author information available on the last page of the article

Vol.:(0123456789)1 3 1028 Human Genetics (2019) 138:1027–1042 domain (NT) and a long cytoplasmic and less conserved (Chung et al. 2007), indicating that any signifcant dysregu- C-terminal domain (CT). Cxs oligomerise in hexameric lation of Gja8 could be deleterious for eye development. complexes called connexons, and allow the transmembrane In humans, missense and frameshift mutations in GJA8 passage of ions and small solutes (≤ 1 kDa). Connexons can (OMIM 600897) have been associated with cataracts (Beyer function independently as hemichannels (HCs) or they can et al. 2013; Yu et al. 2016). Rarely, the phenotype also dock with their counterparts on the juxtaposed cell to form includes additional ocular abnormalities, mainly micro- a gap junction channel (GJC), enabling the direct exchange cornea and iris hypoplasia (Devi and Vijayalakshmi 2006; of small molecules. Given their role in cell–cell communica- Hansen et al. 2007; Hu et al. 2010; Sun et al. 2011; Prokudin tion and tissue homeostasis, Cxs have been implicated in a et al. 2014; Ma et al. 2016), but in a few cases also microph- variety of biological and pathological processes (Pfenniger thalmia (Ma et al. 2016) and sclerocornea (Ma et al. 2018). et al. 2011; García et al. 2016), including myelin-related Interestingly, defects in the formation of the lens have also diseases (Cx32 and Cx47), heart malformations and arrhyth- been observed (Ma et al. 2018). The cataracts described in mia (Cx40), hearing loss and skin disorders (Cx26, Cx30, these individuals vary in both their location (e.g., nuclear, Cx30.3, and Cx31), oculodentodigital dysplasia, a syndrome zonular, lamellar, or total) and appearance (e.g., total, pul- also involving microphthalmia, microcornea, cataract and/ verulent, or dense). The mutations are predominantly het- or spherophakia (Cx43), and early onset cataract (Cx46 and erozygous and only few homozygous variants have been Cx50). reported, all in consanguineous families (Ponnam et al. As with AMC, developmental or early onset cataracts are 2007; Schmidt et al. 2008 Ponnam et al. 2013; Ma et al. a clinically heterogeneous group of disorders, presenting as 2016). These pathogenic variants lead to amino acid altera- isolated anomalies or part of a syndrome. More than 110 tions distributed throughout the protein (Yu et al. 2016), genes have been implicated in congenital cataracts (Gillespie although mostly localised between the domains TM1 and et al. 2014), with mutations in Cxs accounting for around TM2. They are predicted to afect protein function through 16% of cases with a known genetic cause (Shiels and Hejt- various mechanisms, such as by inducing misfolding and/ mancik 2017). Since the lens does not have any blood sup- or mislocalisation or by altering channel properties (Beyer ply, it strongly depends on an extensive network of GJCs et al. 2013). for the intercellular communications that are critical for its Copy number variants (CNVs) in the distal region of development and the maintenance of its transparency. The chromosome 1q21 and including GJA8 are rare in the gen- most abundant Cxs in the lens are Cx46 and Cx50, which eral population, but have recurrently been identifed in indi- can also form mixed hexamers. Cx46, encoded by GJA3, viduals with a broad range of diferent clinical diagnoses is expressed only in fber cells, whereas Cx50, encoded by (Brunetti-Pierri et al. 2008; Meford et al. 2008; Stefansson GJA8, is present throughout the lens. et al. 2008; Bernier et al. 2016). These primarily include Genetic studies in mice have demonstrated that the developmental delay, microcephaly and psychiatric disor- homozygous knockout of either Gja3 or Gja8 leads to cata- ders, although the enrichment of 1q21 CNVs in individuals racts, but with important phenotypic diferences. The dele- with these disorders could partly be related to ascertain- tion of Gja3 causes severe progressive nuclear cataracts, but ment bias. However, some cases also have eye anomalies, does not alter ocular growth (Gong et al. 1997). In contrast, such as cataracts (Brunetti-Pierri et al. 2008; Meford et al. Gja8-null mice develop milder nuclear cataracts at an early 2008; Rosenfeld et al. 2012; Bernier et al. 2016; Ha et al. postnatal age and exhibit signifcantly smaller lenses and 2016), microphthalmia (Meford et al. 2008), and coloboma microphthalmia (White et al. 1998; Rong et al. 2002), indi- (Brunetti-Pierri et al. 2008). Among the genes afected by cating that the two Cxs have overlapping, but distinct func- these recurrent microdeletions/microduplications, GJA8 rep- tions. In addition, the targeted replacement of Gja8 with resents a strong candidate for the ocular anomalies described Gja3 (Cx50KI46 knockin mice) prevents the loss of crys- in some of the 1q21 CNV carriers. talline solubility, but not the postnatal growth defect result- To investigate further the importance of GJA8 in human ing from the Gja8 deletion (White 2002), confrming the eye morphogenesis and provide a better understanding of the functional diversity of the two proteins and the involvement range of developmental ocular anomalies associated with of Gja8 in the control of normal ocular growth. This is also mutations in this gene, we screened GJA8 in a cohort of 426 supported by mouse lines carrying missense mutations in unrelated patients (304 UK, 121 Spanish and 1 large French Gja8 (Steele et al. 1998; Graw et al. 2001; Chang et al. 2002; pedigree) with congenital eye anomalies in the AMC spec- Xia et al. 2012; Berthoud et al. 2013) and by rabbit models trum. Two novel and four known likely pathogenic sequence with CRISPR–Cas9-mediated GJA8 knockout (Yuan et al. variants were identifed in seven families, with one vari- 2016): both develop cataracts, microphthalmia and smaller ant being present in two unrelated families. This expands lenses. Moreover, severe cataracts and small lenses have the catalogue of GJA8 variants likely to be contributing to also been observed in transgenic mice overexpressing Gja8 eye anomalies and the spectrum of phenotypes associated

1 3 Human Genetics (2019) 138:1027–1042 1029 with this gene. Moreover, we also identifed heterozygous 37 Spanish individuals, CNVs were detected from NGS 1q21 microdeletions including the gene GJA8 in fve addi- data using a read depth comparison approach. A detailed tional families, although the pathogenicity of these variants description of the diferent methods can be found in the remains to be established. Supplementary Materials and Supplementary Table 1. The genomic coordinates of the sequence and structural variants are reported according to Build GRCh37/hg19. The allelic Materials and methods frequencies of the sequence mutations were obtained from the Genome Aggregation Database (gnomAD, http://gnoma Cohort description d.broadinstitute.org/) (Lek et al. 2016). For each variant of interest, amino acid conservation across species was visu- A cohort of UK, Spanish and French families with AMC ally inspected using the Vertebrate Multiz Alignment and (Supplementary Table 1) was analysed for GJA8 vari- Conservation (100 Species) track from the UCSC Genome ants. The UK families (n = 304) were recruited as part of Browser. Three conservation scores were annotated using a national ‘Genetics of Eye and Brain anomalies’ study, the database dbNSFP v.3.3 (Liu et al. 2016), specifcally the approved by the Cambridge East Ethics Committee (04/ GERP ++ Rejected Substitutions (RS) score (Davydov et al. Q0104/129), and had not received a genetic diagnosis. Fam- 2010), phyloP 100way_vertebrate score (Siepel et al. 2006), ily 5 was also recruited into the Deciphering Developmen- and phastCons 100way_vertebrate score (Siepel et al. 2005). tal Disorders (DDD) Study, which has UK Research Ethics Putative functional efects of amino acid substitutions were Committee approval (10/H0305/83, granted by the Cam- evaluated with the in silico tools SIFT (Kumar et al. 2009) bridge South REC, and GEN/284/12 granted by the Republic and PolyPhen-2 (Adzhubei et al. 2010). of Ireland REC). The UK families consisted of 55 probands with anophthalmia, 205 with microphthalmia and 44 with Validation of mosaicisms and CNVs other anomalies within the AMC spectrum; 168 individuals were bilaterally afected and 160 had extraocular anomalies. To assess potential mosaicism and independently validate The Spanish families (n = 121) consisted of 6 individuals aCGH fndings, we developed Digital Droplet PCR (ddPCR) with anophthalmia, 42 with microphthalmia and 73 with assays for the sequence variant in family 1 and three of the other anomalies within the AMC spectrum; 100 individuals GJA8 microdeletions identifed (families 12, 13 and 14) were bilaterally afected and 41 had extraocular anomalies. (Supplementary Materials). ddPCR assays were performed They were consented for genetic studies approved by the using a ddPCR QX200 System (Bio-Rad Laboratories). Ethics Committee of the University Hospital Fundación Primers and Taqman probes were specifcally designed for Jiménez Díaz (FJD, Madrid, Spain) and according to the the GJA8 variant p.(Thr39Arg) using a custom Applied Bio- tenets of the Declaration of Helsinki. The four-generation systems TaqMan SNP Genotyping Assay (Thermo Fisher French pedigree consisted of 15 individuals with congenital Scientifc). For CNV analysis, commercial Taqman Copy cataracts and microphthalmia and consented for the study Number assays (Thermo Fisher Scientifc) were used for during their clinical treatment. exon 2 of GJA8 and a reference gene (human RNase P gene).

Identifcation of sequence and structural variants in GJA8 Results

The human gene GJA8 presents one isoform Point mutations identifed in GJA8 (NM_005267.4), comprising of two exons, with the coding sequence (CDS) entirely contained within exon 2. Sequence Screening of the GJA8 coding region in our cohort of 426 variants in the CDS were detected using a combination of individuals with AMC detected 10 missense variants in 11 Next-Generation Sequencing (NGS) methods and direct unrelated families (Tables 1, 2). For each missense variant, sequencing: 35 patients were screened by whole exome the amino acid conservation across species is shown in the sequencing (WES), 207 patients using diferent targeted Supplementary Fig. 1. Taking into account the segregation NGS panels of eye development genes including GJA8, and patterns, the frequency of the variants in public databases 184 patients by Sanger sequencing, which was also used to of unafected individuals (Table 2), in silico predictions validate NGS fndings and check family segregation. In addi- of functional efects and previous reports from the litera- tion, CNV data were available for 188 of these patients: 151 ture (Table 2), as suggested by Richards et al. 2015, six of individuals (96 UK and 55 Spanish) had been assessed by these variants [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), array-based Comparative Genomic Hybridization (aCGH), p.(Phe70Leu), p.(Gly94Arg) and p.(Val97Gly)] were con- with resolutions ranging from 44 to 244 kb, whereas for sidered likely causative, giving a frequency of 1.6% of

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Table 1 Clinical evaluation of the probands carrying GJA8 mutations Family Genetic change Segregation Cataracts Microphthalmia Coloboma Glaucoma Other features

1 NM_005267.4(GJA8):c.116C>G; AD with gonosomal + (B) + (B) − + (L, S) Bilateral corneal p.(Thr39Arg) mosaicism opacifcation, right phthisis, left atrophic optic disc secondary to glaucoma, nystagmus 2 NM_005267.4(GJA8):c.134G>T; AD + (B) + (B) − − Nystagmus p.(Trp45Leu) 3 NM_005267.4(GJA8):c.151G>A; De novo + (B) + (B) − N/A Anterior segment dys- p.(Asp51Asn) genesis, persistent pupillary membrane 4 NM_005267.4(GJA8):c.151G>A; AD + (B) + (B) − − Deep set eyes, corneal p.(Asp51Asn) leukoma, buphthal- mos, corectopia, nystagmus 5 NM_005267.4(GJA8):c.208T>C; AD/de novo in + (B) + (B) − + (S) None p.(Phe70Leu) afected mother 6 NM_005267.4(GJA8):c.280G>A; N/A No lenses + (B) + (B) + (B, P) Congenital aphakia, p.(Gly94Arg) sclerocornea 7 NM_005267.4(GJA8):c.290T>G; De novo + (B) + (B) − + (R, S) Bilateral anterior seg- p.(Val97Gly) ment dysgenesis 8 NM_005267.4(GJA8):c.875T>A; N/A + (B) − + (B) − Nystagmus, photopho- p.(Leu292Gln) bia, small kidneys 9 NM_005267.4(GJA8):c.19C>A; In unafected father − + (R) − − Retinal detachment, p.(Leu7Met) heart anomaly (right double outlet ven- tricle), absent left kidney, septum pel- lucidum abnormality 10 NM_005267.4(GJA8):c.658A>G; In unafected father − + (B) + (B) − Retrobulbar cysts, p.(Asn220Asp) microcephaly associated with normal development, reduced growth 11 NM_005267.4(GJA8):c.997G>C; In unafected father − + (R) + (R) − None p.(Gly333Arg) 12 arr[hg19] 1q21.1q21.2 De novo − − + (B) − Mild dysmorphic (145388977–147395401)x1 features, scoliosis, genu valgum and gastroesophageal refux 13 arr[hg19] 1q21.1q21.2 In unafected father + (R) − + (R) + (S) Detached retina, band (146155983–147824178)x1 keratopathy, long thin fngers and toes 14 arr[hg19] 1q21.1q21.2 N/A − + (R) + (L) − Cleft lip and palate, (146564743–147735011)x1 neonatal seizures 15 arr[hg19] 1q21.1q21.2 De novo − − + (B) N/A Nystagmus (146497694–147825519)x1 16 arr[hg19] 1q21.1q21.2 In afected father − + (R) + (B) N/A Microcephaly (146497694–147825519)x1

AD autosomal dominant, B bilateral, R right eye, L left eye, P primary, S secondary, N/A not available independent individuals with AMC conditions (7/426) car- in the male proband (III:1), who presented with bilateral rying likely pathogenic GJA8 sequence variants. microphthalmia, corneal opacifcation, cataracts and nystag- In family 1 (Fig. 1a), the heterozygous variant mus, left secondary glaucoma and a grossly cupped atrophic p.(Thr39Arg) (NM_005267.4:c.116C>G) was identifed disc (Fig. 1b). Extraocular anomalies were not observed.

1 3 Human Genetics (2019) 138:1027–1042 1031 - - ), Kuo ( 2016 ), Kuo ( 2017 ) et al. ( 2018 ) ( 2016 ) ( 2017 ) ( 2011 ) N/A Novel Ma et al. Ma et al. Novel Ma et al. Ma et al. Novel Ma et al. Ma et al. Mohebi et al. Mohebi et al. Sun et al. Sun et al. N/A References pathogenic pathogenic genic genic genic genic genic genic pathogenic Unlikely Unlikely Uncertain Unlikely Unlikely Likely patho - Likely Likely patho - Likely Likely patho - Likely Likely patho- Likely Likely patho- Likely Likely patho- Likely Unlikely Unlikely Classifcation T (0.68) T (0.3) D (0.01) D (0) D (0) D (0) D (0.02) D (0) D (0) D (0) SIFT (score) T (0) PD (0.976) PD (0.999) PD (0.998) PD (1) PD (1) PD (1) PD (1) PD (1) PD (0.991) Polyphen-2 HDIV (score) 0.202 0.771 1 1 1 1 1 1 1 1 phastCons 2.114 2.235 9.178 6.197 9.92 7.953 8.068 8.068 6.059 2.02 phyloP 2.1 4.71 4.4 5.2 5.2 5.2 5 4.9 4.9 4.25 GERP++_ RS can 0.28% 0.009% N/A 0.24% N/A N/A N/A N/A N/A N/A 0.03%/Afri - Total MAF Total (gnomAD) rs587600450 N/A rs138140155 N/A N/A N/A rs864309703 N/A N/A rs150441169 dbSNP147 identifer CT CT TM4 TM2 TM2 ECL1 ECL1 TM1 TM1 NT Domain p.(Gly333Arg) p.(Leu292Gln) p.(Asn220Asp) p.(Val97Gly) p.(Gly94Arg) p.(Phe70Leu) p.(Asp51Asn) p.(Trp45Leu) p.(Thr39Arg) p.(Leu7Met) Aa change Aa c.997G>C c.875T>A c.658A>G c.290T>G c.280G>A c.208T>C c.151G>A c.134G>T c.116C>G c.19C>A cDNA change cDNA sequence variants identifed in this variants study sequence GJA8 chr1:147381079 chr1:147380957 chr1:147380740 chr1:147380372 chr1:147380362 chr1:147380290 chr1:147380233 chr1:147380216 chr1:147380198 chr1:147380101 2 Table cDNA positions refer to NM_005267.4, the protein changes refer to NP_005258.2. Protein domains: NT, N-terminal domain; TM, transmembrane domain; ECL, extracellular loop; CT, C-ter loop; CT, N-terminal domain; ECL, extracellular domain; TM, transmembrane domains: NT, NP_005258.2. Protein to refer changes NM_005267.4, the to protein positions refer cDNA minal domain. For each variant, dbSNP147 identifers and total minor allele frequencies from gnomAD are reported (N/A, information not available). Conservations scores (GERP++_RS, scores Conservations available). reportedare gnomAD not from information (N/A, frequencies allele minor total and identifers dbSNP147 variant, each For domain. minal algorithm the the score, the each conserved more site. GERP++_RS For the larger the from database dbNSFP v.3.3. retrieved were and phastCons100way_vertebrate) phyloP100way_vertebrate conser PhastCons a minimum of − 20.0 from a maximum of 10.003 in dbNSFP. to range scores p values) (phylogenetic a minimum of − 12.3 from a maximum of 6.17. PhyloP to range scores of GERP++_RS scores ≥ 2, values considered PhyloP ≥ 2 and PhastCons ≥ 0.9 1. We of the 0 to In silico predictions as conserved. reported from functional efects are range scores for vation tolerated) damaging; T: and SIFT (D: damaging; PD: probably Polyphen-2 - Genomic coordi nates (hg19)

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Fig. 1 GJA8 likely pathogenic sequence variants identifed in ▸ seven unrelated families with AMC. a Pedigree of family 1. Sanger sequencing results showing the segregation of the missense variant p.(Thr39Arg) are presented. The chromatogram of individual II:2 is suggestive of mosaicism. b Photographs of the afected individuals of family 1 showing intra-familial phenotypic variability. The mother (II:2, top) had a milder phenotype with normal sized eyes, bilateral pseudophakia following surgery for early onset cataract and right exotropia. The image of the proband (III:1, bottom) shows his bilateral microphthalmia, complete corneal opacifcation and right phthisis. The proband was also diagnosed with cataracts, nystagmus and left atrophic disc secondary to glaucoma. c Absolute quantifcation of the allele abundance for the variant c.116C>G; p.(Thr39Arg) in family 1. Digital Droplet PCR (ddPCR) assays were performed using a Taqman FAM-labeled probe for genotyping the mutant allele and a VIC-labeled probe to detect the wild-type allele. On the left, 1D fuorescence amplitude plot of droplets shows mutant allele detection in the FAM channel for the heterozygous carrier (III:1), the putative mosaic mother (II:2), a wild-type homozygous carrier (I:2) and no template control (NTC). FAM-positive droplets (blue), containing the mutant allele, exhibit increased fuorescence compared to negative droplets (grey). On the right, the fractional abundance of the mutated allele, represented in percentage, was calculated for the FAM-positive droplets versus VIC-positive droplets (wild-type allele), confrming the mosaicism of this variant in individual II:2. d Pedigree of family 2. On the left, a representative sequence chromatogram shows the heterozygous missense variant p.(Trp45Leu). The genotype of the six individuals tested for the variant is indicated below each symbol. e–f Pedigree of families 3 and 4, both carrying the missense variant p.(Asp51Asn). The sequence chromatograms show that the variant occurred de novo in family 3. N/A, genotype not available. In family 4, representative sequence chromatogram showing the p.(Asp51Asn) and pedigree indicating the inheritance of afected status and of the variant. For family 4, fully flled symbols represent individuals afected with congenital cataracts and microphthalmia, but without glaucoma, quarter flled symbols represent individuals with congenital glaucoma. g Pedigree of family 5 and sequence chromatograms showing the missense variant p.(Phe70Leu). h Pedigree of family 6. The adopted child carries the missense variant p.(Gly94Arg). i Pedi- gree of family 7. Sanger sequencing results show that the missense variant p.(Val97Gly) arose de novo in the child II:1

The Sanger sequencing profle was suggestive of mosaicism in his mother (II:2), who was diagnosed with early onset cataracts and right exotropia. Mosaicism was confrmed and quantifed by ddPCR in blood samples, with an estimated fractional abundance of 25% for the mutated allele (Fig. 1c). The variant was absent in the maternal grandparents, suggesting that it arose as a de novo post-zygotic event in the mother. The substitution of threonine 39, located in the TM1 domain, is predicted deleterious by Polyphen-2 and SIFT. Interestingly, the change p.(Thr39Arg) is absent in dbSNP147 and gnomAD, but has been previously described in a family with congenital cataracts, microcornea and iris hypoplasia (Sun et al. 2011). In family 2 (Fig. 1d), a four-generation pedigree with autosomal dominant congenital cataracts, we initially identifed the variant p.(Trp45Leu) (NM_005267.4:c.134G>T) in the proband III:2, who had a diagnosis of dense congenital cataracts, microphthalmia and nystagmus. Sanger

1 3 Human Genetics (2019) 138:1027–1042 1033 sequencing was performed on fve additional family mem- In family 5 (Fig. 1g), the novel variant p.(Phe70Leu) bers (four afected with the same clinical diagnosis and one (NM_005267.4:c.208T>C) was identifed in the proband unafected) and showed that the variant co-segregated with III:2, diagnosed with bilateral microphthalmia, congenital the ocular phenotype. The same amino acid substitution, cataracts, and secondary glaucoma. Segregation analysis predicted deleterious by Polyphen-2 and SIFT, has been showed that the mutation was a de novo event in the afected previously described in another multi-generation family mother (II:2), who also had microphthalmia and cataracts. including 11 individuals with autosomal dominant congeni- Phenylalanine 70 is a conserved amino acid located in the tal cataracts (Mohebi et al. 2017). Moreover, a diferent mis- ECL1 domain, and its substitution is predicted to be deleteri- sense variant afecting the same amino acid, p.(Trp45Ser) ous by SIFT and Polyphen-2. (NM_005267.4:c.134G>C, rs864309688), has been reported In family 6 (Fig. 1h), we identifed a missense variant in a three-generation family with bilateral congenital cata- p.(Gly94Arg) (NM_005267.4:c.280G>A) in a male proband racts and microcornea (Vanita et al. 2008), in a sporadic of Chinese ethnicity (II:1) presenting with bilateral congeni- case with bilateral anterior cortical/nuclear cataracts (Ma tal aphakia (absence of the lens), corneal opacity, bilateral et al. 2016), and in a three-generation family with paediat- microphthalmia with iris and optic disc coloboma, and ric cataracts (Javadiyan et al. 2017). Functional experiments bilateral primary glaucoma. No extraocular anomalies were showed that p.(Trp45Ser) inhibited the formation of func- observed. No details of parental phenotype or DNA were tional intercellular channels or hemichannels and decreased available. Interestingly, this change, predicted deleterious the junctional conductance induced by wild-type Cx50 and by SIFT and Polyphen-2 and located in the TM2 domain, is Cx46, acting as dominant negative inhibitor (Tong et al. absent in gnomAD, but has been previously identifed as a 2011). Tryptophan 45 is an evolutionary conserved residue de novo event in a child with bilateral corneal opacifcation located in the TM1 domain and its substitution with leucine and microcornea, bilateral rudimentary lenses and bilateral or serine has not been observed in controls (gnomAD). glaucoma (Ma et al. 2018). In family 3 (Fig. 1e) and family 4 (Fig. 1f), we identi- In family 7 (Fig. 1i), the female proband (II:1) carried a fed the variant p.(Asp51Asn) (NM_005267.4:c.151G>A; de novo variant p.(Val97Gly) (NM_005267.4:c.290T>G), rs864309703), which afects a highly conserved amino acid predicted deleterious by SIFT and Polyphen-2 and located located in the ECL1 domain. This change, predicted deleteri- in the TM2 domain. This previously undescribed variant was ous by Polyphen-2 and SIFT, has been previously reported in identifed by the DDD study (DECIPHER ID: 259194) and a patient with bilateral microphthalmia, congenital cataracts confrmed with Sanger sequencing. She had bilateral micro- and sclerocornea (Ma et al. 2016, 2018). In family 3, the phthalmia, anterior segment dysgenesis and dense cataracts, mutation occurred as a de novo event in the male proband, treated with lensectomies, and right secondary glaucoma, who presented with bilateral microphthalmia with associated with no extraocular features. cataracts, anterior segment dysgenesis, and persistent pupil- The significance of the other four variants identified lary membranes. Extraocular anomalies were not observed. in the screening (Supplementary Fig. 2) was considered In the three-generation family 4, the heterozygous variant ‘uncertain’ [p.(Leu292Gln)] or ‘unlikely to be pathogenic’ was identifed in both the proband (III:1) and her afected [p.(Leu7Met), p.(Asn220Asp), and p.(Gly333Arg)]. father (II:4). Head axial computed tomography scanning of A novel amino acid change, p.(Leu292Gln) the proband at 29 years showed borderline bilateral micro- (NM_005267.4:c.875T>A), was identifed in a proband with phthalmia and enophthalmos (posterior displacement of the bilateral mild cataracts and optic nerve coloboma associated eye), although her ocular globes had a size of 20 mm (right with nystagmus, photophobia, and small kidneys (family 8). eye) and 18 mm (left eye). At 32 years of age, the proband The substitution of leucine 292, located in the CT domain, had no light perception on the right and light perception is predicted benign by SIFT, but deleterious by Polyphen-2. on the left. The right eye was phthisical, with no discern- Sanger sequencing of PAX2 revealed that the proband II:3 ible anterior segment structures; the left eye had a corneal also carried a novel heterozygous frameshift variant in this leukoma, cataract and corectopia. The father was diagnosed gene [NM_003987.2:c.529_530ins13, p.(Ala177Glyfs*8)], with bilateral microphthalmia and congenital cataracts. The which introduces a premature stop codon in exon 5. Sanger paternal grandfather (I:1) and one of the paternal uncles sequencing excluded the maternal inheritance of both the (II:3), now deceased, were also afected. The mother (II:5) GJA8 and the PAX2 variants; paternal DNA was unavailable was afected by congenital glaucoma. However, the proband for segregation analysis. did not carry a mutation in any known congenital glaucoma- The GJA8 variant p.(Leu7Met) associated genes included in a custom-targeted NGS panel (NM_005267.4:c.19C>A; rs150441169), located in the containing 121 eye developmental genes, 9 of which are N-terminal domain and predicted deleterious by SIFT associated with congenital glaucoma. No extraocular anoma- and Polyphen-2, was detected in a patient (II:1) with syn- lies were observed. dromic unilateral microphthalmia, and was inherited from

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◂Fig. 2 GJA8 structural variants identifed in fve unrelated families change occurs in the CT domain and is predicted as tolerated with AMC. a Modifed schematic from the UCSC Genome Browser by both SIFT and Polyphen-2. (NCBI Build GRCh37/hg19). Partial ideogram of the chromosome bands 1q21.1–q21.2 and the multiple blocks of highly homologous segmental duplications (SDs) present in this region are shown. SDs, 1q21 copy number variants overlapping with GJA8 reported under the UCSC track ‘Duplications of > 1000 Bases of Non-RepeatMasked Sequence’, are stretches of DNA of at least 1 kb GJA8 is part of a complex genomic locus, 1q21.1–q21.2, in length, sharing a sequence identity of at least 90% with another genomic region on the same or on a diferent chromosome (inter- or characterised by the presence of numerous segmental dupli- intra-chromosomal SD). The colours indicate diferent levels of simi- cations (SDs), which make the region susceptible to recur- larity between duplications (grey: 90–98% similarity, yellow: 98–99% rent rearrangements. To investigate whether structural vari- similarity, orange: greater than 99% similarity). The breakpoint ants afecting GJA8 were present in our cohort of families regions (BP2, BP3, and BP4) overlapping with these SD clusters are represented by green bars. The genomic locations of the 1q21 dele- with AMC, we examined a subset of 188 unrelated individu- tions identifed in this study are represented by red bars and indicated als for whom copy number information was available from with family identifers. RefSeq Genes are indicated by dark-blue rec- aCGH and/or NGS data. As such, the samples for which tangular bars. For genes with multiple isoforms, the bars represent the CNV data were generated were not chosen according to any coordinates of the maximal region among the isoforms. b Pedigrees of the families carrying heterozygous 1q21 deletions selection criteria applied across the total cohort, and therefore they efectively represented a randomly-selected subset of independent AMC cases. This resulted in the identifca- his unafected father (family 9). The family is of Afri- tion of 1q21 microdeletions in fve families (Fig. 2, Table 1). can ethnicity and the minor allele frequency (MAF) for The frst microdeletion was identifed in a proband (fam- the African/African–American population in gnomAD ily 12, Fig. 2b) with bilateral coloboma of the iris and cho- is 0.28%. Diferent substitutions of this amino acid have roid, mild dysmorphic features (broad forehead, narrow been described before as disease-causative mutations: palpebral fssures, depressed nasal root, and low set ears), p.(Leu7Pro) (NM_005267.4:c.20T>C) was identifed in scoliosis, genu valgum and gastroesophageal refux. She a family with inherited cataracts (Mackay et al. 2014) and had normal developmental milestones. This CNV, detected p.(Leu7Gln) (NM_153465.1:c.20T>A) in a rat model with from the screening of a custom NGS panel of 121 eye devel- nuclear pulverulent cataracts and, in the case of homozy- opment genes, was further confrmed by both aCGH and gous rat mutants, microphthalmia with hypoplastic lens ddPCR (Supplementary Fig. 3). It spans approximately (Liska et al. 2008). However, in contrast with these previ- 2 Mb (chr1:145388977–147395401, Build GRCh37/hg19) ously reported variants, the frequency of the p.(Leu7Met) and afects 40 RefSeq genes. Segregation analysis revealed variant in unafected individuals, in particular of African/ that this structural variant arose as a de novo event in the African-American ethnicity suggests that the substitution proband. with a methionine might be tolerated. The second microdeletion was identified in a female The variant p.(Asn220Asp) (NM_005267.4:c.658A>G; proband with unilateral chorioretinal coloboma involving rs138140155, gnomAD total MAF = 0.24%) was identifed the optic disc, band keratopathy, cataract, and secondary in an individual with bilateral microphthalmia and chorioret- glaucoma without extraocular anomalies, and was inher- inal colobomas involving the optic disc, as well as micro- ited from her unafected father (family 13, Fig. 2b). The cephaly associated with normal development and faltering minimal deleted region (chr1:146155983–147824178, Build growth (family 10) and was inherited from her unafected GRCh37/hg19) spans approximately 1.67 Mb and afects father. This substitution of asparagine 220, located in the 24 RefSeq genes. To validate the microdeletion and test the TM4 domain and predicted deleterious by SIFT and Poly- hypothesis that the unafected status of the father could be phen-2, has been reported before in a proband with con- due to mosaicism, we performed a ddPCR assay. However, genital cataract and microcornea (Ma et al. 2016) and in a this experiment confrmed the full heterozygous status of the three-generation family with congenital cataracts and apha- microdeletion in both individuals (Supplementary Fig. 3b). kic glaucoma (Kuo et al. 2017). However, in those families, The third microdeletion was found in a female proband it did not co-segregate with the phenotype and, therefore, with extreme microphthalmia in the right eye and iris, cho- was classifed as benign. This was also supported by func- rioretinal coloboma in the left eye, cleft lip and palate, and tional experiments showing that this rare polymorphism did neonatal seizures (family 14, Fig. 2b). The minimal deleted not abolish intercellular channel function (Kuo et al. 2017). region (chr1:146564743–147735011, Build GRCh37/hg19) The variant p.(Gly333Arg) (NM_005267.4:c.997G>C; spans approximately 1.17 Mb and afects 17 RefSeq genes. rs587600450, gnomAD total MAF = 0.009%) was observed The presence of the CNV in the mother was excluded by in a proband with unilateral microphthalmia and chorioreti- ddPCR (Supplementary Fig. 3b). The father and other family nal coloboma involving the optic disc, and was inherited members were unavailable for phenotypic and segregation from her unafected father (family 11). This heterozygous analysis.

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The fourth microdeletion (chr1:146497694–147825519, of the Pax6-dependent regulatory circuit and contributes to Build GRCh37/hg19), spanning approximately 1.33 Mb and mouse lens formation (Cantù et al. 2014). No other genes afecting 20 RefSeq genes, was identifed in two independ- known to be relevant in eye development are present in the ent families (families 15 and 16, Fig. 2b). In family 15, it region. occurred as a de novo event in a proband with bilateral iris Sequence analysis of the coding region of GJA8 and chorioretinal coloboma involving disc, and nystagmus, in the probands carrying 1q21 microdeletions did not without extraocular anomalies. The presence of the CNV in reveal any variant on the remaining allele. In family 12, the parents was excluded by aCGH. Clinical re-assessment no additional pathogenic variants were identified from of the family revealed that the father had blue dot lens opaci- the targeted NGS screening of 121 eye developmen- ties and cavernous disc anomalies with a pit in the right eye tal genes. In family 13, the NGS targeted sequencing of and mild cavernous disc anomaly or pronounced optic cup 351 diagnostic genes for eye developmental anomalies in the left eye. In family 16, a three-generation pedigree in proband II:2 identifed an in-frame deletion of 6 bp in with coloboma, the microdeletion was detected by aCGH in FOXC1 [NM_001453.2:c.1338_1343del, p.(Gly447_ the proband (III:6), who showed bilateral chorioretinal colo- Gly448del)], maternally inherited. This rare variant (gno- boma and microphthalmia in the right eye associated with mAD total MAF = 0.06%) is reported as a multi-allelic microcephaly and normal development, and in the afected SNP (rs572346201), which occurs in a region coding for father (II:5), who presented with microphthalmia and colo- a poly-glycine stretch. Although its clinical signifcance is boma in the right eye. The cousin III:2 was also afected with unknown, due to the repetitive nature of this region, it is unilateral iris and chorioretinal coloboma. However, segre- likely to represent a natural polymorphism. gation analysis could not be performed on this individual. The predicted boundaries of these CNVs indicated that these rearrangements belonged to diferent classes of 1q21 Discussion microdeletions. Recurrent 1q21 CNVs occur at four breakpoint regions (BP1–BP4), each corresponding to a large Mutations in Cx50, encoded by GJA8, have been primarily block of highly homologous SDs (Meford et al. 2008). Fur- linked to congenital and early onset cataract in humans and thermore, the locus can be divided into two distinct regions: also animal models. However, recently in a small number a proximal region included between BP2 and BP3 and a of cases GJA8 mutations have also been associated with a distal region, fanked by BP3 and BP4, which mediate the broader phenotype which can include microphthalmia, scle- most recurrent CNVs of the 1q21 locus. While the microde- rocornea and lens abnormalities (Ma et al. 2018). letions found in families 13–16 were distal rearrangements In this study, we have investigated the role of GJA8 in occurring between BP3 and BP4 (class I), the microdeletion a cohort of 426 individuals with a wide range of devel- detected in family 12 was fanked by the breakpoints BP2 opmental eye anomalies, and identifed 16 families with and BP4 and extended from the proximal through to the AMC carrying genetic alterations of GJA8. These included distal region (class II). Despite their rarity in the general six likely pathogenic sequence variants [p.(Thr39Arg), population, both types of 1q21 microdeletions appeared to p.(Trp45Leu), p.(Asp51Asn), p.(Phe70Leu), p.(Gly94Arg), be enriched in our AMC cohort. We compared the frequency and p.(Val97Gly)] detected in seven unrelated families, of these CNVs in our cases with control individuals pre- four missense variants [p.(Leu7Met), p.(Asn220Asp), viously reported in the literature (Rosenfeld et al. 2012): p.(Leu292Gln), and p.(Gly333Arg)] with uncertain or BP3–BP4 microdeletions occurred in 4 out of 188 individu- unlikely clinical signifcance and four heterozygous 1q21 als with AMC versus 12 out of 65,282 controls (2.13% ver- microdeletions involving GJA8, detected in fve unrelated sus 0.02%, Fisher’s exact test p = 1.17 × 10−7), whereas families, of uncertain signifcance. BP2–BP4 microdeletions occurred in 1 out of 188 individu- Segregation analyses were possible for fve out of six als with AMC versus 1 out of 65,927 controls (0.532% ver- likely pathogenic sequence variants and showed that these sus 0.002%, Fisher’s exact test p = 0.0057). occurred either de novo or co-segregated with the disease Taking into account all the 1q21 microdeletions iden- in an autosomal dominant fashion. These variants were tifed in our cohort, the minimally deleted region spans bioinformatically predicted damaging and have not been approximately 830 kb (chr1:146564743–147395401, Build reported in unafected individuals according to public data- GRCh37/hg19) and includes 11 genes (NBPF19, NBPF13P, bases. Interestingly, three of these changes [p.(Thr39Arg), PRKAB2, CHD1L, PDIA3P1, FMO5, LINC00624, BCL9, p.(Trp45Leu) and p.(Asp51Asn)] have been previously ACP6, GJA5, and GJA8). In addition to Gja8, a role in eye described in families with cataracts (Sun et al. 2011; Ma development has been shown also for Bcl9, a co-activator et al. 2016; Javadiyan et al. 2017; Mohebi et al. 2017; Ma for β-catenin-mediated transcription in Wnt signaling (Bienz et al. 2018) and a fourth [p.(Gly94Arg)] in a proband with 2005). A recent study has demonstrated that Bcl9 is also part sclerocornea and lens abnormalities (Ma et al. 2018). Given

1 3 Human Genetics (2019) 138:1027–1042 1037 the rarity of these variants, the identifcation of the same motifs for regulatory kinases (Liu et al. 2011; Wang et al. missense changes in unrelated afected individuals strongly 2013). In this region, we identifed a novel missense change, supports a causative relationship between these variants and p.(Leu292Gln), of unknown clinical signifcance. The vari- eye developmental disorders. In particular, p.(Asp51Asn) ant was found in a proband (family 8) who also carried an had been reported as a de novo mutation in a patient with insertion of 13 bp in PAX2 (NM_003987.2:c.529_530ins13, bilateral microphthalmia, congenital cataracts, and scle- p.(Ala177Glyfs*8)). Heterozygous variants of PAX2 (MIM rocornea (Ma et al. 2016). In the present study, the same 167409) are identifed in approximately half of the cases pre- variant was detected in two independent families with a senting with renal coloboma syndrome (Bower et al. 2012), similar phenotype, including microphthalmia, cataracts, and also known as papillorenal syndrome (OMIM 120330). other anterior chamber eye anomalies. This emerging geno- Therefore, this novel PAX2 variant is likely to be responsi- type–phenotype correlation suggests that this amino acid ble for optic nerve coloboma and kidney anomalies observed substitution might have a severe efect on GJA8 function and in the patient, but it is possible that the GJA8 variant might supports the involvement of this protein in a broader range lead to a subtle efect on the protein function and contribute of eye developmental anomalies. to his mild cataract phenotype. Our identifcation of the variant p.(Gly94Arg) in another By contrast, the three additional heterozygous variants patient also aids genotype–phenotype correlation for amino p.(Leu7Met) (family 9), p.(Asn220Asp) (family 10) and acid substitutions of this highly conserved residue. In our p.(Gly333Arg) (family 11) were considered as likely benign. cohort, the change was identifed in a case with bilateral These were identifed in individuals with AMC, but with- corneal opacifcation, congenital aphakia and microphthal- out cataracts, in unafected parents either in this or previous mia with iris and optic disc coloboma. The same variant has studies (Ma et al. 2016; Kuo et al. 2017) and in controls been previously reported as a de novo event in an individual (gnomAD). diagnosed with bilateral corneal opacifcation, glaucoma, Human GJA8 maps to a structurally complex locus on and rudimentary lenses (Ma et al. 2018). Interestingly, Ma chromosome 1q21.1–q21.2, with at least four large blocks of et al. (2018) also described another variant afecting the highly homologous SDs, which make it prone to nonallelic same amino acid, p.(Gly94Glu), in a proband with total scle- homologous recombination (NAHR) (Meford et al. 2008). rocornea and cataractous disc-like lenses with microcornea. As with other genomic loci subject to recurrent rearrange- Mice models expressing heterozygous missense mutations ments (such as 15q11, 15q13, 16p11.2, 16p12.1, 16p13.11, (e.g. Cx50D47A, Cx50S50P, Cx50V64A, and Cx50R205G) 17q12, and 22q11.2) (Girirajan and Eichler 2010; Stankie- (Graw et al. 2001; Xia et al. 2006, 2012; Berthoud et al. wicz and Lupski 2010), 1q21 CNVs have been associated 2013) or with complete Gja8 knockout (White et al. 1998; with a wide range of phenotypes including dysmorphic Rong et al. 2002) have shown that Gja8 is important for features, developmental delay, neuropsychiatric disorders, lens development. Therefore, the identifcation of glycine and cardiac and eye anomalies. The reported eye anoma- 94 mutations in three individuals with lens abnormalities lies include cataracts (Brunetti-Pierri et al. 2008; Meford supports the hypothesis that this amino acid is particularly et al. 2008; Rosenfeld et al. 2012; Bernier et al. 2016; Ha important for GJA8 to perform this role in eye development. et al. 2016) and in a minority of cases more severe defects Interestingly, the phenotype of bilateral aphakia associated such as microphthalmia (Meford et al. 2008) and coloboma with sclerocornea overlaps with that of individuals with bial- (Brunetti-Pierri et al. 2008). lelic mutations in FOXE3 (Iseri et al. 2009). Therefore, when The most common 1q21 CNVs occur between the break- screening patients with this phenotype, it is important to points BP3 and BP4 (Fig. 2a), spanning ~ 1.35 Mb (Mef- screen for variants in both FOXE3 and GJA8. ford et al. 2008). This region contains only ~ 800 kb of Multiple sequence alignment indicated that all likely path- unique (i.e., nonduplicated) DNA sequence (Bernier et al. ogenic sequence variants identifed in our cohort afected 2016) and includes at least 11 genes (NBPF19, NBPF13P, conserved residues (Supplementary Fig. 1) and were located PRKAB2, CHD1L, PDIA3P1, FMO5, LINC00624, BCL9, within the N-terminal region of the protein (Fig. 3). Our ACP6, GJA5, and GJA8), which might contribute to difer- fndings are consistent with the previous studies, since muta- ent aspects of the disease manifestations observed. Alter- tions associated with cataracts tend to cluster between TM1 natively, 1q21 CNVs can involve only the proximal region and TM2 (Yu et al. 2016). The transmembrane domains are (BP2–BP3) or both the proximal and the distal region (BP1/ thought to play an important role in oligomerisation and BP2–BP4). Microdeletions of the proximal region have been pore formation, while the ECL1 domain is important in reported to be a predisposing factor for thrombocytopenia- the docking of two opposing HCs to form the GJCs. While absent radius (TAR) syndrome (Klopocki et al. 2007), these domains are evolutionarily conserved and present together with sequence variants in the RBM8A gene. Within high homology among the members of the Cx family, the the distal region, a potential role in eye development has CT region is the most isotype-specifc domain and contains been shown for two of the genes, GJA8 and BCL9. A recent

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Fig. 3 GJA8 mutation spectrum. Schematic of GJA8 showing the genic, blue, likely benign and grey, unknown clinical signifcance. NT protein domains according to UniProt (entry ID: P48165). Above: N-terminal domain, TM transmembrane domain, ECL extracellular previously published mutations are shown. Below: the missense vari- loop, ICL cytoplasmic loop, CT C-terminal domain ants identifed in our cohort are indicated: red indicates likely patho- study has demonstrated that Bcl9 is a downstream efec- frequency in individuals with clinical diagnoses compared tor of Pax6 during mouse lens development (Cantù et al. with controls (Brunetti-Pierri et al. 2008; Meford et al. 2014). However, the role of BCL9 in human eye develop- 2008; Rosenfeld et al. 2012; Bernier et al. 2016). In par- ment has not yet been established. Given the involvement of ticular, the comparison of a large cohort of individuals with GJA8 in both cataractogenesis and ocular growth, as previ- developmental delay, intellectual disability, dysmorphic fea- ously described, this gene seems to be a good candidate for tures and congenital anomalies with previously published the ocular anomalies observed in some of the 1q21 CNV control cohorts showed that the frequency of BP2–BP4 carriers. deletions was 0.024% in cases (11/45,744) versus 0.002% In most cases, the 1q21 rearrangements are inherited. in controls (1/65,927), whereas the frequency of BP3–BP4 Their presence in unafected parents has brought into ques- deletions was 0.285% in cases (86/30,215) versus 0.018% tion their pathogenic signifcance, but the analysis of large in controls (12/65,282) (Rosenfeld et al. 2012). This enrich- clinical and population cohorts has shown that 1q21 micro- ment suggests that these CNVs might increase susceptibil- deletions/microduplications occur at signifcantly higher ity to developmental anomalies with variable expressivity

1 3 Human Genetics (2019) 138:1027–1042 1039 and incomplete penetrance, although the factors underly- the heterozygous change presented with bilateral cataracts ing their heterogeneous phenotypes remain unexplained. In and microphthalmia, his mother, who was 25% mosaic for this study, we identifed four microdeletions in fve families, this variant, had a milder phenotype of early onset cata- three overlapping with the microdeletions/microduplications racts. Therefore, we hypothesize that the somatic mosaicism recurrently found between breakpoints BP3 and BP4 and detected in the mother may explain her milder phenotype one larger BP2–BP4 microdeletion encompassing both the and that lower doses of aberrant GJA8 protein during eye proximal and the distal region. Given the rarity of these development might be responsible for less severe phenotypic rearrangements, the presence of 1q21 microdeletions in outcomes. In support of this, a recent study has reported our AMC cohort, with a frequency of 2.13% in AMC cases a correlation between the severity of developmental eye for BP3–BP4 microdeletions (p = 1.17 × 10−7) and a fre- abnormalities and somatic mosaicisms of Pax6 mutations quency of 0.53% in AMC cases for BP2–BP4 microdeletions in CRISPR/Cas9 genome-edited mouse embryos (Yasue (p = 0.0057), seems to support their role as a risk factor for et al. 2017). developmental disorders, including eye anomalies. However, In conclusion, this study expands our knowledge of the consistent with the previous studies, the segregation pat- role of GJA8 in eye development, highlighting how genetic tern in families 13 and 15 indicates that other genetic and/ alterations of this gene are likely to give rise not only to or environmental modifers are likely to be important for the early onset cataracts, but also to other developmental eye phenotypic outcome. Therefore, the exact genotype–pheno- anomalies. The screening of GJA8 in 426 individuals with type correlation remains to be established. AMC resulted in the identifcation of six likely pathogenic Mouse models have shown that Gja8 copy number losses variants in seven families. In the six families where segrega- and point mutations act through different mechanisms tion analysis was possible, the variants co-segregated with and modes of inheritance. Deletions of the entire coding both early onset cataracts and microphthalmia. In one sin- region of the gene cause cataracts and microphthalmia only gleton case with aphakia and corneal opacifcation, where no when homozygous, indicating a recessive mode of inher- segregation analysis was possible, we identifed the variant itance (White et al. 1998; Rong et al. 2002). In contrast, p.(Gly94Arg). This fnding, in combination with two previ- mouse strains carrying pathogenic Gja8 missense muta- ously reported patients with lens development abnormalities tions develop microphthalmia and cataracts in a dominant and with variants afecting the same amino acid, highlights or semi-dominant fashion (Steele et al. 1998; Graw et al. the importance of this specifc residue in the function of 2001; Chang et al. 2002; Liska et al. 2008; Xia et al. 2012). GJA8 and suggests that GJA8 mutations can be responsible Since Cxs function in hexameric complexes which can be for phenotypes often associated with FOXE3 variants. The homo- or heteromeric, it is possible that the impact of single role of GJA8 microdeletions in AMC remains uncertain: amino acid substitutions may be more severe than the loss the enrichment of rare 1q21 microdeletions in our cohort of one functional allele. Mutant Cx subunits can interfere seems to support their role as risk factors for developmental with correct formation of the oligomeric complexes in a eye disorders. However, the incomplete segregation and the dominant negative manner and, since GJCs can be formed phenotypic variability of these variants indicate that other by diferent types of Cx subunits, this efect can also extend genetic and/or environmental factors might be of impor- to the function of other Cxs. Functional and cellular stud- tance. In summary, these data expand the spectrum of human ies have shown that point mutations can alter the activity phenotypes associated with GJA8 variants and the identifca- of the human GJA8 protein in various ways (Beyer et al. tion of specifc mutations contributes to our understanding 2013). For instance, pathogenic variants can cause misfold- of their genotype–phenotype correlation. Therefore, this ing, improper oligomerisation and/or trafcking defects, study demonstrates the importance of screening GJA8 in leading to a reduced number of functional channels on the individuals with developmental eye anomalies. membrane. Alternatively, the pathogenic variants could alter some physiological properties of the channels, such Acknowledgements We would like to thank the patients and their families for their participation in our study. We are grateful to Suzanne as permeability or conductance, or lead to the formation of Broadgate for assisting with the coordination of the project. We thank HCs with new and aberrant functions. Therefore, a single the Centrum Menselijke Erfelijkheid laboratory, Leuven, Belgium, for base mutation can afect several aspects of the Cx function. the diagnostic PAX2 testing. We also thank Dr Jean-Philippe Bault for This complexity may explain the phenotypic heterogeneity the ultrasound examinations performed on family 2. This work was GJA8 supported by grants from Baillie Giford, Visually Impaired Children observed among the carriers of variants, and also the Taking Action (VICTA) (http://www.victa.org.uk/), Microphthal- diference in penetrance between sequence and copy number mia, Anophthalmia, Coloboma Support (MACS) (http://www.macs. variations. org.uk), Oxford Brookes University Central Research Fund, Retina Intra-familial phenotypic variability was also observed for France, Fondation Maladies Rares, Spanish Institute of Health Carlos III (CP12/03256), Spanish Ministry of Economy and Competitiveness the sequence variant identifed in family 1 [p.(Thr39Arg)] (SAF2013–46943-R), Mutua Madrileña Foundation and Cátedra de possibly related to mosaicism. While the proband carrying

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Patrocinio UAM-IIS-FJD of Genomic Medicine (University Autónoma under selective constraint using GERP++. PLoS Comput Biol of Madrid). The DDD study presents independent research commis- 6(12):e1001025 sioned by the Health Innovation Challenge Fund [Grant number Devi RR, Vijayalakshmi P (2006) Novel mutations in GJA8 associ- HICF-1009-003], see http://www.ddduk.org/access.html for full ated with autosomal dominant congenital cataract and micro- acknowledgement. cornea. Mol Vis 12:190–195 Fernandez-San Jose P, Corton M, Blanco-Kelly F, Avila-Fernandez Compliance with ethical standards A, Lopez-Martinez MA, Sanchez-Navarro I, Sanchez-Alcudia R, Perez-Carro R, Zurita O, Sanchez-Bolivar N et al (2015) Targeted next-generation sequencing improves the diagnosis of Conflict of interest On behalf of all authors, the corresponding author autosomal dominant retinitis pigmentosa in Spanish patients. states that there is no confict of interest. 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Afliations

1 Faculty of Health and Life Sciences, Oxford Brookes 11 Wessex Regional Genetics Laboratory, Salisbury NHS University, Gipsy Lane, Oxford OX3 0BP, UK Foundation Trust, Salisbury, UK 2 Genetics Service, Instituto de Investigación 12 Shefeld Clinical Genetics Department, Northern General Sanitaria-Fundación Jiménez Díaz University Hospital, Hospital, Shefeld, UK Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, 13 Wessex Clinical Genetics Service, Princess Anne Hospital, Spain University Hospital Southampton NHS Foundation Trust, 3 Centre for Biomedical Network Research on Rare Diseases Southampton, UK (CIBERER), ISCIII, Madrid, Spain 14 Human Genetics and Genomic Medicine, Southampton 4 Service de Génétique Médicale, Hôpital Purpan, CHU General Hospital, University of Southampton, Southampton, Toulouse, Toulouse, France UK 5 UMR 1056 Inserm, Université de Toulouse, Toulouse, 15 Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK France 16 Cheshire and Merseyside Genetics Service, Liverpool 6 Oxford Centre for Genomic Medicine, Oxford University Women’s NHS Foundation Trust, Liverpool, UK Hospitals NHS Foundation Trust, Oxford, UK 17 Department of Clinical Genetics, Centre de Référence 7 Institute of Ophthalmology, University College London, “AnDDI Rares”, Poissy Hospital GHU PIFO, Poissy, France London, UK 18 Fondation Ophtalmologique Adolphe-de-Rothschild, Paris, 8 Medical Genetics Department, University Hospital Virgen de France la Arrixaca, Murcia, Spain 19 Clinical Genetics Unit, St Georges University of London, 9 West Midlands Regional Genetics Laboratory, Birmingham London, UK Women’s and Children’s NHS Foundation Trust, Birmingham, UK 10 West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women’s and Children’s NHS Foundation Trust, Mindelsohn Way, Birmingham B15 2TG, UK

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